Transforming growth factor-beta (TGF-β) is abundant in bone matrix and has been shown to regulate the activity of osteoblasts and osteoclasts in vitro and in vivo. Human Adipose derived Mesenchymal Stromal Cells (ASC) are precursors of osteoblasts, adipoblasts and chondroblasts. Thus, studies initially focused on the secretion of cytokines by ASC which have a profound effect in bone remodeling, such as Tgf-ß1, Osteoprotegerin (OPG) and Hepatocyte Growth Factor (HGF).
TGF-β1 concentrations are high in subchondral bone from humans with osteoarthritis. High concentrations of TGF-β1 induced formation of nestin-positive mesenchymal stem cell (MSC) clusters, leading to formation of marrow osteoid islets accompanied by high levels of angiogenesis (Zhen G, et al. (Nat Med. 19: 704-12, 2013). It has been found that transgenic expression of active TGF-β1 in osteoblastic cells induced osteoarthritis, whereas inhibition of TGF-β activity, by means of a TßRII dominant negative receptor, in subchondral bone, attenuated the degeneration of articular cartilage leading to less development of osteoarthritis. It has also been reported that mice which express a dominant negative type II TGF-β receptor (TßRII-DN) in osteoblasts, show decreased TGF-β responsiveness in osteoblasts and increased bone volume, demonstrating that endogenous TGF-beta acts directly on osteoblasts to regulate bone remodeling, structure and biomechanical properties (Filvaroff, E. et al. Development, 126: 4267-4279, 1999). In addition, TGF-β also regulates osteoclastogenesis and osteoclast survival, in part through the induction of osteoprotegerin (OPG), a protein known to inhibit osteoclast formation and function (Thirunavukkarasu K, et al. J. Biol. Chem. 276:36241-36250, 2001).
Transgenic mice that overexpress the dominant-negative type II TGF-β receptor (dnTgfbr2) in skeletal tissue exhibit progressive skeletal degeneration (Buckwalter J A, et al. Clin Orthop Relat Res 423: 7-16, 2004). The articular chondrocytes in the superficial zone of cartilage tissue become hypertrophic with increased type X collagen expression. Loss of proteoglycan and progressive degradation of cartilage tissue have been observed in 6-month-old mice which strongly resemble human osteoarthritis (OA) (OA-like) (Serra R, et al. J Cell Biol 139: 541-552, 1997). TGF-β signaling plays a critical role not only in the regulation of chondrocyte homeostasis during cartilage destruction, but also in the manipulation of subchondral bone cell behavior during osteophyte formation, another feature of OA (van der Kraan P M, et al. Osteoarthr Cartilage 15: 237-244, 2007).
The role of the TGF-β signaling pathway in osteophyte formation was further explored by blocking studies using specific TGF-β inhibitors. Several groups demonstrated that ablation of endogenous TGF-β activity, by intra-articular overexpression of soluble TGF-β type II receptor extracellular domain or Smad7, suppresses osteophyte formation in experimental murine OA models (Scharstuhl A, et al. J Immunol 169: 507-514, 2002). These observations clearly demonstrate that TGF-β plays a dominant role in the induction of osteophytes, at least in murine OA models.
In vivo, TGF-β1 also induces angiogenesis (Madri J A, et al. J Cell Biol. 106: 1375-1384, 1988; Roberts A B, Proc Natl Acad Sci USA. 83: 4167-4171, 1986; Yang E Y, et al. J Cell Biol. 111: 731-741, 1990.). In OA, high TGF-β1 levels are also accompanied by high levels of angiogenesis. Hepatocyte growth factor (HGF) is a potent mitogen, morphogen, and motogen for a variety of cells, mainly epithelial cells. Increased expression of the HGF/HGF-receptor system in osteoarthritic cartilage, suggest a regulatory role in the homeostasis and pathogenesis of human joint cartilage (Pfander D, et al. Osteoarthritis Cartilage. 7: 548-59, 1999).
Previous studies have shown that TGF-β can promote angiogenesis and tumor invasion via stimulation of HGF expression (Chu S H, et al. J NeurooncoL, 85: 33-38, 2007; Lewis M P, et al. Br J Cancer 90: 822-832, 2004)). Conversely, TGF-β has also been shown to inhibit HGF transcription, potentially through binding of a TGF-β inhibitory element located approximately 400 bp upstream of the HGF transcription start site (Liu Y, et. al. J Biol Chem., 269: 4152-4160, 1994; Plaschke-Schlütter A, et al. J Biol Chem., 270: 830-836, 1995), and abrogation of this effect leads to cancer development (Cheng N, et al. Cancer Res. 67: 4869-4877, 2007).
Quinolones (QNs) antibiotics such as Ciprofloxacin (CPFX) were widely used in clinical practice owing to their wide spectrum antibacterial activity and high degree of bioavailability. They were not approved for use in children and adolescents due their toxic effects on joint cartilage of immature animals (Cuzzolin L, et al. Expert Opin Drug Saf 1: 319-24, 2002). Quinolones, administered systemically, caused arthropathy and tendinopathy when given during the growth phase (Sendzik J, et al. Int J Antimicrob Agents 33: 194-200, 2009.). It was reported that Ciprofloxacin decreased thickness of articular cartilage of the femoral condyle, inhibit proliferation of cultivated chondrocytes and secretion of soluble proteoglycans in a concentration- and time-dependant manner in juvenile rats (Li, P. et al. Arch. Pharmacol. Sin. 25: 1262-1266, 2004).
Chondrocyte cluster formation is a feature of all mechanical and chemical OA models (Moriizumi T, et al. Virchows Arch B Cell Pathol Incl Mol Pathol., 51: 461-474, 1986; van der Kraan P M, et al. Am J Pathol., 135:1001-1014, 1989). Animals with quinolone arthropathy showed cavities in the middle zone of the articular cartilage containing necrotic chondrocytes. After 14 days, many of the lacunae in defective areas contained chondrocyte clusters. When treated for 14 days, and after a 14-day recovery period, territorial matrix had been deposited around individual chondrocytes within the clusters, indicating that in immature joints there is a certain degree of spontaneous repair by cluster cells (Sharpnack D D, et al. Lab Anim Sci., 44: 436-442, 1994). It has been shown that TGF-β1 is activated in the subchondral bone in response to altered mechanical loading in an anterior cruciate ligament transection (ACLT) osteoarthritis mouse model (Zhen G, et al. Nat Med. 19: 704-12, 2013). Additionally, CPFX was found to up-regulate TGF-β1 production by HT-29 cells and its anti-proliferative effect was abolished when TGF-β1 was blocked (Bourikas L A, et al. Br J Pharmacol. 157: 362-70, 2009).
Adipose derived stem cells (hASCs) express cytokines such as IL-6, GM-CSF and Flt3-ligand (Tholpady S S, et al. Clin Plast Surg 33: 55-62, 2006; Katz A J, et al. Stem Cells. 23: 412-23, 2005; Schafer A, et al. Stem Cells 25: 818-827, 2007). These cytokines are regulated by TGF-β1 either negatively (GM-CSF, SCF and Flt3-ligand) (Jacobsen S E, et al. J Immunol., 151: 4534-4544, 1993; Jacobsen S E, et al. Blood 87: 5016-5026, 1996) or positively (IL-6, TPO) (Ramsfjell V, et al. J Immunol. 158: 5169-5177, 1997.). Recently, overexpression of a dominant negative mutant of the human TβRII receptor (TβRII-DN) in mammalian cells has been shown to be very effective in blocking TGF-β1 action. This mutant, based on the isoform A of the receptor, is capable to bind TGF-β1 but signaling is disrupted due to the absence of a serine/threonine kinase domain. TβRIIA-DN has been shown to disrupt TGF-β1 mediated signaling allowing the study of the behavior of different cell types in the absence of either a paracrine or an autocrine effect of the cytokine (Fan X, et al. The Journal of Immunology 168: 755-762, 2002.).
Various documents disclosing different TGF-β1 receptors, chimerics, fusion proteins, domains, are known, for example, EP0975771, WO 2008/157367, US 2006/0247198, U.S. Pat. No. 6,001,969, and WO 94/09815.